Our overall goal is to elucidate the neuronal mechanisms that control mitochondria to satisfy the energy demands of nerve terminals. Mitochondria accumulate within nerve terminals where they generate most of the ATP required for the release and recycling of neurotransmitters. Neural function, therefore, relies on mitochondria generating sufficient ATP to sustain neurotransmitter release. Similarly, mitochondria power presynaptic Ca2+ homeostasis. A failure in neuronal Ca2+ homeostasis has catastrophic consequences and is a hallmark of many neurodegenerative diseases. Surprisingly, we know very little about the mechanisms that coordinate mitochondrial number and function with presynaptic energy requirements, yet understanding these mechanisms will be critical to understanding the progression of neurodegenerative disease. Our central hypothesis is that neuronal mechanisms control the number and function of mitochondria to accommodate presynaptic energy requirements, and that these mechanisms are synapse specific. We propose to elucidate these mechanisms in the musculoskeletal system of the fruit fly larva, where each motor neuron terminal has a different work rate which we can quantify using electrophysiological and Ca2+-imaging techniques. Diversity in presynaptic energy requirements, genetic tractability and accessibility to neurophysiological techniques, make this an ideal system in which to investigate neuronal mechanisms that control mitochondria to accommodate presynaptic energy requirements. In Aim 1, we will determine whether mitochondria are supplied to motor nerve terminals in numbers that are proportional to their work rate. 3D-EM reconstruction will be used to determine mitochondrial number. We will also probe the relationship between mitochondrial number and function. In Aim 2, we will test the hypothesis that mitochondrial volume is controlled at the level of different terminals on the same axon. Mitochondrial functional parameters will be determined at individual terminals to test whether mitochondrial function may be different between terminals on the same axon. In Aim 3, we will test the hypothesis that, over the course of development, active zone spacing and bouton diameter adjust to the firing rate of the motor neuron to bring presynaptic Ca2+ levels into a range most effective at stimulating mitochondrial energy metabolism during presynaptic activity. In so far as Ca2+ regulation is a heavy consumer of presynaptic ATP, this in situ model of presynaptic bioenergetics will provide an essential context for a better understanding of the early events involving mitochondrial dysfunction and Ca2+ dysregulation in neurodegenerative disease.